Methods of inhibiting cataracts and presbyopia

ABSTRACT

Described herein are methods of inhibiting or reversing the progression of cataract formation or presbyopia in an eye by administering a γ-crystallin charge masking agent. Both presbyopia and cataracts are caused by aggregation of the soluble crystalline lens proteins called the crystallins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US2014/027852, filed on Mar. 14, 2014,which claims the benefit of priority to U.S. provisional application No.61/782,860, filed on Mar. 14, 2013, under the provisions of 35 U.S.C.119 and the International Convention for the protection of IndustrialProperty, are incorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to methods of inhibiting or reversing theprogression of age related changes in the crystalline lens of an eye.

BACKGROUND

The crystalline lens of the eye is a transparent structure that issuspended immediately behind the iris, which brings rays of light to afocus on the retina. The lens contains both soluble and insolubleproteins; together they constitute 35 percent of the wet weight of thelens. In a young, healthy lens, the soluble proteins, commonly referredto as crystallins, constitute 90 percent of the lens proteins. Duringthe aging process, the lens crystallins form insoluble aggregates,which, at least in part, account for the decreased deformability of thelens nucleus, which characterizes presbyopia, the loss of the eye'sability to change focus to see near objects. The formation of insolubleaggregates of lens crystallins in presbyopia is believed to be an earlystage in the formation of age-related cataracts.

Cataracts are defined by cloudiness or opacification in the crystallinelens of the eye. As an individual ages, cataracts form as thecrystallins present in the lens are converted into aggregates, resultingin increased lens opacity. Specifically, there is a progressive decreasein the concentration of the soluble chaperone, α-crystallin, in humanlens nuclei with age, as it becomes incorporated into high molecularweight aggregates and insoluble protein. The presence of aggregatescompromises the health and function of the lens and left untreated,cataracts can lead to substantial vision loss or even blindness.Presently, the most common treatment for cataracts is surgery.

Crystallins are structural proteins most highly expressed in the lensfiber cells of the vertebrate eye. The crystallins are divided into twosubfamilies: the α-crystallins (αA and αB) which are members of thesmall heat shock protein superfamily, also functioning as structuralproteins and molecular chaperones; and the evolutionarily-linkedsuperfamily of β- and γ-crystallins which function primarily asstructural proteins in the lens, and contribute to the transparency andrefractive properties of lens structure. In addition to their role incataract development, αA-crystallin and αB-crystallin have beenimplicated in neurodegenerative diseases, like Alexander's disease,Creutzfeldt-Jacob disease, Alzheimer's disease and Parkinson's disease.

U.S. Patent Application 2008/0227700 describes deaggregation of proteinsusing peptides having chaperone activities as a therapeutic treatment.Specifically, αB peptides were used to deaggregate pH-induced aggregatesof β-crystallin as measured by light scattering. Provision of acontinuous supply of alpha crystallins into the lens is a challenge.What is needed are alternative methods suitable for the deaggregation ofcrystallins for the inhibition and/or reversal of cataracts andpresbyopia.

SUMMARY

In one aspect, a method of inhibiting or reversing the progression ofcataract formation or presbyopia in an eye comprises contacting the eyewith an effective cataract or presbyopia-inhibiting amount of anophthalmic composition comprising at least one γ-crystallin chargemasking agent, wherein the charge masking agent is not a polypeptide.

In another aspect, an ophthalmic composition comprises a bifunctionalmolecule containing a leaving group covalently linked to a molecularbristle.

In another aspect, a method of inhibiting or reversing the progressionof age related degeneration of a crystalline lens in an eye comprisescontacting the eye with an effective degeneration-inhibiting amount ofan ophthalmic composition comprising at least one γ-crystallin chargemasking agent, wherein the γ-crystallin charge masking agent is not apolypeptide.

In yet another aspect, a method of treating a disease relating toprotein folding in a patient in need thereof comprises administering atherapeutically effective amount of a bifunctional molecule containing aleaving group covalently linked to a molecular bristle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows MALDI-TOF data for modification of

-crystallin with PEG24.

FIG. 2 shows the NICF of

-crystallin modified with PEG24.

FIG. 3 shows the distribution function of

-crystallin modified with PEG24.

FIG. 4 shows the graph of Γ versus q² for

-crystallin modified with PEG24.

FIG. 5 shows MALDI-TOF data for modification of

-crystallin with PEG4.

FIG. 6 shows the NICF of

-crystallin modified with PEG4.

FIG. 7 shows the distribution function of

-crystallin modified with PEG4.

FIG. 8 shows the graph of Γ versus q² for

-crystallin modified with PEG4.

FIG. 9 shows MALDI-TOF data for modification of

-crystallin with CAPEG.

FIG. 10 shows the NICF of

-crystallin modified with CAPEG4.

FIG. 11 shows the distribution function of

-crystallin modified with CAPEG4.

FIG. 12 shows the graph of Γ versus q² for

-crystallin modified with CAPEG4.

FIG. 13 shows the distribution function of

-crystallin modified with MMPEG.

FIG. 14 shows the distribution function of

-crystallin modified with Biotin.

FIG. 15 shows the distribution function of

-crystallin modified with BiotinPEG.

FIG. 16 shows the distribution function of

-crystallin modified with sulfo-N-hydroxysuccinimide acetate.

FIG. 17 shows embodiments of charge masking groups. In each structure, Ris the molecular bristle.

FIG. 18 shows embodiments of molecular bristles.

FIG. 19 shows a schematic of the measurement of trans-epithelialtransport.

FIG. 20 shows the CAPEG4 that was transported to the bottom of the cellin the tran-epithelial transport experiment.

FIG. 21 shows the pH of Opisol® versus the mg/ml of added CAPEG4.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Disclosed herein are methods of disaggregating/preventing formation of aγ-crystallin aggregate comprising contacting the γ-crystallin aggregatewith a composition comprising a γ-crystallin charge masking agent in anamount sufficient to disaggregate and/or prevent formation of theγ-crystallin aggregate. One of ordinary skill in the art would recognizethat while the molecules disclosed herein are described as aγ-crystallin charge masking agents they may also disaggregate/preventprotein aggregation of β-crystallin as well. Further disclosed aremethods of inhibiting or reversing the progression of cataract formationin an eye which comprises contacting the eye with an effectivecataract-inhibiting amount of an ophthalmic composition comprising aγ-crystallin charge masking agent. Also disclosed are methods ofinhibiting or reversing the progression of presbyopia in an eye whichcomprises contacting the eye with an effective presbyopia-inhibitingamount of an ophthalmic composition comprising a γ-crystallin chargemasking agent. In specific embodiments, the γ-crystallin charge maskingagent is not a polypeptide.

The inventors herein have employed techniques such as dynamic lightscattering to study the aggregates formed by γ-crystallins in solution.Both the β and γ-crystallins are highly stable structural proteinscomprising four Greek-key motifs in two domains. While the β-crystallinsform dimers as well as hetero- and homo-oligomers, the γ-crystallins aremonomers in the eye. Further, while the β-crystallins exhibit arepulsive force in solution, the γ-crystallins exhibit an attractiveinteraction attributed to nonspecific protein or water interactions. Ithas also been hypothesized that thiol modifications cause aggregates ofγ-crystallin to form in solution.

The human γ-crystallin family contains five members, the γA-Dcrystallins and γ-S crystalline. The γA-D crystallins are expressedearly in development and are primarily found in the lens core; γC andγD-crystallin are most prevalent. Unfolding and refolding of γ-Dcrystalline in vitro has been shown to lead to increased proteinaggregation due to the lack of stability of the refolded protein.γS-crystallin has been shown to be a key protein in the suppression ofaggregation of other crystalline proteins, leading to a clear lens.

Without being held to theory, it is believed that the aggregation ofγ-crystallin is both an electrostatic and hydrophobic phenomenon, withthe electrostatic forces dominating. Adding the heat shock proteins αA-and αB-crystallin disrupts γ-crystallin aggregation. A γ-crystallincharge masking agent that can disrupt electrostatic interactions cansubstitute for the chaperone activity of α-crystallin and prevent/reduceγ-crystallin aggregate size.

Treatment with γ-crystallin charge masking agents can be used to treatdiseases and/or conditions resulting from aggregation of γ-crystallinssuch as cataracts and presbyopia. As used herein, a cataract is anopacity of the crystalline lens of the eye caused by altered proteininteractions in the lens. Protein interactions include misfolding ofproteins as well as protein-protein interactions such as aggregation.Presbyopia is the impairment of vision due to advancing years or oldage. Symptoms of presbyopia include decreased focusing ability for nearobjects, eyestrain, difficulty reading fine print, fatigue while readingor looking at an illuminated screen, difficulty seeing clearly up close,less contrast when reading print, need for brighter and more directlight for reading, needing to hold reading material further away inorder to see it clearly, and headaches, especially headaches when usingnear vision. Individuals suffering from presbyopia may have normalvision, but the ability to focus on near objects is at least partiallylost over time, and those individuals come to need glasses for tasksrequiring near vision, such as reading. Presbyopia affects almost allindividuals over the age of 40 to a greater or lesser degree.

In the method of inhibiting the progression of cataract formation in aneye, the eye may already contain one or more developing or fullydeveloped cataracts before it is contacted with the γ-crystallin chargemasking agent. Accordingly, the method can be used to inhibit theformation of further cataracts in the eye, or to inhibit the formationof mature cataracts from the developing cataracts already present in theeye. Alternatively, the eye may be free of any developing or fullydeveloped cataracts before it is contacted with the γ-crystallin chargemasking agent.

In the method of reversing the progression of cataract formation in aneye, at least partial to full reversal of cataracts in the eye isachieved by contacting the eye with a γ-crystallin charge masking agentas disclosed herein.

Similarly, in the method of inhibiting the progression of presbyopia inan eye, the individual may already be experiencing one or more symptomsof presbyopia before the eye is contacted with the γ-crystallin chargemasking agent. Accordingly, the method can be used to reduce theprogression of the symptom(s) experienced, or to inhibit the formationof additional symptoms of presbyopia. Alternatively, the eye may be freeof any symptoms of presbyopia before it is contacted with theγ-crystallin charge masking agent.

In the method of reversing the progression of presbyopia in an eye, atleast partial to full reversal of the symptoms of presbyopia in the eyeis achieved by contacting the eye with a γ-crystallin charge maskingagent as disclosed herein.

As used herein, γ-crystallin charge masking agent is a molecule suitableto interfere with γ-crystallin electrostatic protein-proteininteractions which lead to γ-crystallin aggregation. In one embodiment,the masking agent is not a polypeptide. γ-crystallin charge maskingagents prevent γ-crystallin aggregates from forming and/or reduce thesize of pre-formed aggregates.

In one embodiment, the γ-crystallin charge masking agent is a highconcentration salt solution, having a salt concentration over 400 mM.The term “salt” as used herein, is intended to include an organic orinorganic salt, including but not limited to one or more of NaCl, KCl,ammonium halides such as NH₄Cl, alkaline earth metal halides such asCaCl₂, sodium acetate, potassium acetate, ammonium acetate, sodiumcitrate, potassium citrate, ammonium citrate, sodium sulphate, potassiumsulphate, ammonium sulphate, calcium acetate or mixtures thereof.Additional organic salts include alkylammonium salts such asethylammonium nitrate, sodium citrate, sodium formate, sodium ascorbate,magnesium gluconate, sodium gluconate, trimethamine hydrochloride,sodium succinate, and combinations thereof. Without being held totheory, it is believed that the identity of the ion, e.g., Li+, Na+ andK+, can affect the ability of the γ-crystallin charge masking agent toprevent γ-crystallin aggregation.

It was unexpectedly shown herein that salt, e.g. KCl, concentrations ofless than 300 nM did not provide a reduction in the size of γ-crystallinaggregates. However, at KCl concentrations to 400 to 1000 mM, aggregateformation was effectively inhibited.

In another embodiment, the γ-crystallin charge masking agent is abifunctional molecule containing a leaving group covalently linked to amolecular bristle. The bifunctional molecule interacts with charges onthe γ-crystallin molecules, such as positively charged lysine andarginine residues and negatively charged glutamate and aspartateresidues. The molecular bristle is a hydrophilic, water-soluble speciesthat provides distance between the γ-crystallin molecules, preventingaggregation. Without being held to theory, it is believed that thebifunctional molecule reacts and effectively puts the molecular bristleonto the protein, and the leaving group is expelled during the reaction.The covalently attached molecular bristle prevents aggregation of theγ-crystallin molecules. Without being held to theory, it is believedthat the bifunctional molecules described herein may also act asβ-crystallin interaction inhibitors.

Exemplary leaving groups (also called reactive groups) includesuccinimide and carboxylic acid functional groups, specificallyN-hydroxysuccinimide and COOH. In some embodiments, the leaving group isbiocompatible. Other examples of leaving groups include isocyanate,isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide,anhydride, fluorobenzene, carbonate, N-hydroxysuccinimide ester,imidoester, epoxide, and fluorophenyl ester. FIG. 17 shows embodimentsof leaving groups wherein the R group is the molecular bristle. When thebifunctional molecule contains COOH, water leaves when it reacts with aprotein's amine group. When the bifunctional molecule containsN-hydroxysuccinimide water is not released. In an NH₂ reaction, waterleaves when NH₂ reacts with a COOH group on the protein.

Exemplary molecular bristles include linear or branched polyethyleneglycols having 4 or more oxyethylene groups, such as 4 to 200oxyethylene groups. Also included are modified polyethylene glycols suchas alkoxy- and aryloxy polyethylene glycols having 4 to 200,specifically 4 to 24 oxyethylene, alkloxy ethylene or aryloxy ethylenegroups. Alternative molecular bristles includepoly(2-hydroxypropyl)methacrylamide (HPMA),poly(2-hydroxyethyl)methacrylate (HEMA), poly(2-oxazilines),poly(m-phosphocholine), poly lysine, and poly glutamic acid. FIG. 18shows embodiments of molecular bristles. In one embodiment, themolecular bristle has a number average molecular weight of 150 to 8000.

In a specific embodiment, the bifunctional γ-crystallin charge maskingagent is:

In one embodiment, the bifunctional γ-crystallin charge masking agentsdescribed herein are also useful in the treatment of diseases relatingto protein folding such as Alzheimer's disease, Parkinson's disease, andHuntington's disease. In a specific embodiment, the bifunctionalγ-crystallin charge masking agents are administered as oralcompositions.

In one embodiment, the γ-crystallin charge masking agent is not apolypeptide. “Polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

An advantage of the γ-crystallin charge masking agents described hereinis that they are expected to be effective in the presence ofpost-translational modifications of γ-crystallins, including, forexample, transamidation, oxidation, modified and dysfunctionalconnexins, and high concentrations of inorganic and organic ions such asCa⁺².

The γ-crystallin charge masking agents are contacted with the eye toinhibit the progression of cataracts and/or reduce existing cataracts,or to inhibit and/or reduce the symptoms of presbyopia. As used herein,the term “contacting the eye” encompasses methods of directly applyingthe γ-crystallin charge masking agent to the eye. In the above-describedmethod, suitable means known to those of ordinary skill in the art maybe used to contact the eye with the compound. Examples of such methodsinclude, but are not limited to, the compound being injected into theeye, being dropped or sprayed into the eye, applied in the form of anophthalmic device, applied by iontophoresis, or otherwise topicallyapplied to the eye.

As used herein, the term “effective cataract-inhibiting amount” means anamount which will inhibit the progression or formation of cataracts inan eye or inhibit the progression or formation of mature cataracts fromdeveloping cataracts already present in the eye. The effectivecataract-inhibiting amount of the γ-crystallin charge masking agent willdepend on various factors known to those of ordinary skill in the art.Such factors include, but are not limited to, the size of the eye, thenumber and progression of any fully developed or developing cataractsalready present in the eye, and the mode of administration. Theeffective cataract-inhibiting amount will also depend on whether thepharmaceutical composition is to be administered a single time, orwhether the pharmaceutical composition is to be administeredperiodically, over a period of time. The period of time may be anynumber of days, weeks, months, or years. In one embodiment, theeffective cataract-inhibiting amount of the γ-crystallin charge maskingagent, specifically the bifunctional molecules described herein, isabout 0.001 g to about 0.1 g. Specifically, the effectivecataract-inhibiting amount is about 0.01 g to about 0.05 g.

As used herein, the term “effective presbyopia-inhibiting amount” meansan amount which will reduce a symptom of presbyopia in an eye or inhibitthe progression of additional symptoms of presbyopia in the eye. Theeffective presbyopia-inhibiting amount of the γ-crystallin chargemasking agent will depend on various factors known to those of ordinaryskill in the art. Such factors include, but are not limited to, the sizeof the eye, the number and type of symptoms already present in theindividual, and the mode of administration. The effectivecataract-inhibiting amount will also depend on whether thepharmaceutical composition is to be administered a single time, orwhether the pharmaceutical composition is to be administeredperiodically, over a period of time. The period of time may be anynumber of days, weeks, months, or years. In one embodiment, theeffective presbyopia-inhibiting amount of the γ-crystallin chargemasking agent, specifically the bifunctional molecules described herein,is about 0.001 g to about 0.1 g. Specifically, the effectivepresbyopia-inhibiting amount is about 0.01 g to about 0.05 g.

As used herein the term “ophthalmic composition” refers to apharmaceutically acceptable formulation, delivery device, mechanism orsystem suitable for administration to the eye. The term “ophthalmiccompositions” includes but are not limited to solutions, suspensions,gels, ointments, sprays, depot devices or any other type of formulation,device or mechanism suitable for short term or long term delivery ofβ_(L)-crystallin electrostatic interaction inhibitors to the eye. Incontrast to oral formulations, for example, ophthalmic compositionsexhibit specific technical characteristics associated with theirapplication to the eyes, including the use of pharmaceuticallyacceptable ophthalmic vehicles that avoid inducing various reactionssuch as, for example, irritation of the conjunctiva and cornea, closureof the eyelids, secretion of tears and painful reactions. Specificophthalmic compositions are advantageously in the form of ophthalmicsolutions or suspensions (i.e., eye drops), ophthalmic ointments, orophthalmic gels containing β_(L)-crystallin electrostatic interactioninhibitors. Depending upon the particular form selected, thecompositions may contain various additives such as buffering agents,isotonizing agents, solubilizers, preservatives, viscosity-increasingagents, chelating agents, antioxidizing agents, antibiotics, sugars, andpH regulators.

Examples of preservatives include, but are not limited to chlorobutanol,sodium dehydroacetate, benzalkonium chloride, pyridinium chlorides,phenethyl alcohols, parahydroxybenzoic acid esters, benzethoniumchloride, hydrophilic dihalogenated copolymers of ethylene oxide anddimethyl ethylene-imine, mixtures thereof, and the like. Theviscosity-increasing agents may be selected, for example, frommethylcellulose, hydroxyethylcellulose, carboxymethylcellulose,hydroxypropylmethylcellulose, polyvinyl alcohol, carboxymethylcellulose,chondroitin sulfate, and salts thereof. Suitable solubilizers include,but are not limited to, polyoxyethylene hydrogenated castor oil,polyethylene glycol, polysorbate 80, and polyoxyethylene monostearate.Typical chelating agents include, but are not limited to, sodiumedetate, citric acid, salts of diethylenetriamine pentaacetic acid,diethylenetriamine pentamethylenephosphonic acid, and stabilizing agentssuch as sodium edetate and sodium hydrogen sulfite.

Exemplary buffers include, but are not limited to borate buffers,phosphate buffers, carbonate buffers, acetate buffers and the like. Theconcentration of buffer in the ophthalmic compositions may vary fromabout 1 mM to about 150 mM or more, depending on the particular bufferchosen.

As used herein, the term “vehicle” is intended to include a carrier,diluent or excipient suitable for ophthalmic use. “Excipient” refers toan ingredient that provides one or more of bulk, imparts satisfactoryprocessing characteristics, helps control the dissolution rate, andotherwise gives additional desirable characteristics to thecompositions. In particular, the excipients are selected such that theophthalmic composition does not trigger a secretion of tears that willentrain the active ingredient. Acceptable excipients are well known to aperson skilled in the art, who will know how to select them depending onthe desired formulation.

In one embodiment, the γ-crystallin charge masking agent is administeredin the form of an ophthalmic device, such as a contact lens or a punctalplug. Suitable ophthalmic devices included biocompatible devices with acorrective, cosmetic or therapeutic quality.

In one embodiment, the γ-crystallin charge masking agent may be adheredto, incorporated into or associated with a contact lens, optionally as acontrolled-release composition. The contact lens may be produced usingthe known materials, for example hydrogels, silicone hydrogels, siliconeelastomers and gas permeable materials such as polymethylmethacrylate(PMMA), methacrylic acid ester polymers, copolymers ofoligosiloxanylalkyl(meth)acrylate monomers/methacrylic acid and thelike. Specific examples of materials for water-containing softophthalmic lenses include those described in U.S. Pat. No. 5,817,726,2-hydroxyethyl methacrylate polymers as described in U.S. Pat. No.5,905,125, ophthalmic lens materials as described in European PatentApplication No. 781,777, the hydrogel lens which is coated with a lipidlayer in advance as described in U.S. Pat. No. 5,942,558; allincorporated herein for their teachings regarding contact lenses.Generally used contact lens such as hard or rigid cornea-type lens, andgel, hydrogel or soft-type lens which are produced from the above knownmaterials may be used.

It is common in the contact lens industry to characterize contact lensesinto two major categories; conventional and silicone hydrogels. Theconventional based hydrogels started as poly(hydroxyethyl methacrylate)(poly HEMA) and evolved to polyHEMA copolymers with other hydrophilicmoieties such as n-vinyl pyrrolidone (nVP), acrylamide, dimethylacrylamide, and methacrylated phosphorylcholines. Polyvinyl alcohollenses may also be employed.

The silicone hydrogels (SiH) typically consist of copolymers ofmethacrylated or meth(acrylamide) silicone monomers, prepolymers ormacromers with typical conventional hydrogel monomers. Examples ofsilicone monomers include “Tris”, alkyl terminated, methacrylatedpolydimethylsiloxane (PDMS), and block copolymers of silicone andhydrophilic monomers. ABA triblock copolymers are common where the Agroup is a hydrophilic block and the B group is the silicone monomerblock. In addition to the methacrylates, other reactive groups includevinyl, acrylamide, or any other reactive group capable of chain reactionpolymerization. Crosslinking and polymerization can also be achieved viastep-growth polymerization using monomers with bi-functionality. Anexample is the reaction of a hydroxyl group with a carboxylic acid groupin two amino acids or from terepthalic acid and ethylene glycol.

Plasma based coating methods are commonly used on silicone hydrogelsincluding plasma oxidation and plasma coatings.

A sustained-release γ-crystallin charge masking agent composition may beproduced, for example, by incorporating in, associating with or adheringto the contact lens the γ-crystallin charge masking agent compositionaccording to the known methods for producing the contact lenses withsustained-release drugs as described in U.S. Pat. Nos. 5,658,592;6,027,745; WO2003/003073; US-2005-0079197, incorporated herein for theirteachings regarding contact lenses and sustained release. Specifically,the contact lens may be produced by adhering the γ-crystallin chargemasking agent to a part of a finely-divided or gel sustained-releasingagent such as polyvinyl pyrrolidone, sodium hyaluronate and the like. Inaddition, sustained release may be produced by forming a γ-crystallincharge masking agent composition reservoir such as by producing acontact lens from a member which forms a front surface of the lens and amember which forms a rear surface of the lens.

In one embodiment the charge masking agent may be inserted into theaqueous or vitreous as an injection with controlled release.

In one embodiment, the γ-crystallin charge masking agent is administeredin a punctal plug. As used herein, the term punctal plug refers to adevice of a size and shape suitable for insertion into the inferior orsuperior lacrimal canaliculus of the eye through, respectively, theinferior or superior lacrimal punctum.

In one embodiment, the γ-crystallin charge masking agent is administeredby iontophoresis. Iontophoresis is a technique using a small electriccharge to deliver a medicine or other chemical through the skin.

In one embodiment, the ophthalmic composition is administered usingultrasound enhancement.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Cloning of γ-Crystallin

The

D- and

S-crystallin DNA sequences are in pQe1 plasmids that were provided bythe King Labs at Massachusetts Institute of Technology (Cambridge,Mass.).

-crystallin protein sequences contain a 6×N-terminal histidine tag (histag) for purification purposes. The plasmids were transformed into acloning competent cell line to create additional plasmid DNA. Plasmidswere subsequently transformed into an expression competent cell line forprotein synthesis (TAM 1 E. coli cells (Active Motif. Carlsbad,Calif.)).

-crystallin plasmid DNA was chemically transformed into M15pRep E. colicells for protein synthesis. 1 L cultures were grown for proteinpurification.

-crystallin protein was purified by Ni affinity chromatography. TheN-terminal His tag contained on the

-crystallin proteins preferentially binds to the Ni column. The boundprotein can be eluted with an imidazole gradient which competitivelybinds to the Ni, releasing the purified protein. Purity was confirmed bySDS PAGE gel electrophoresis and fast protein liquid chromatography(FPLC).

Example 2: Effect of pH and Salt on Purified D- and S-Crystallin

Because pH and salt have an effect on the aggregation of β-crystallin,the effect of pH and salt on γ-crystallin was also studied. Particlesizes were measured using dynamic light scattering (DLS).

Experimental dynamic light scattering (DLS) was measured using an ALVgoniometer instrument which had an ALV-5000/E correlator equipped with288 channels (ALV, Langen Germany) and a 2 W argon laser (Coherent Inc.,Santa Clara, Calif.), with a working power of approximately 40 mW.Scattering intensity was measured at angles between 30° and 90° at 5°intervals, corresponding to a scattering wave vector (q) range between8.41×10⁶ and 2.30×10⁷ m⁻¹. The scattering wave vector is defined asq=4πn sin (θ/2)/λ, where θ is the scattering angle, and λ=514.5, thewavelength of the argon laser in vacuum, and n is 1.33, the refractiveindex of water. The temperature of the sample was held at constanttemperature of 4 to 37±0.1° C. by a circulating water bath.

The correlation function was analyzed using CONTIN analysis to calculatelag times for the correlation functions at measured angles. The peaksrepresent the diffusive mode of the proteins. The delay times (τ) wereconverted to Γ and graphed versus q². The slope of the fitted line isthe diffusion coefficient (D). The lines are fitted such that theintercept is zero because a non-zero value of q=0 is unphysical. The Γversus q² plots were linear with r² values of 0.95 or greater.

DLS measurements were performed on both modified and unmodified

D- and

S-crystallin proteins at 150 mM NaCl, 20 mM Na₂HPO₄/NaH₂PO₄ buffer pH6.8 at a concentration of 0.5 mg/mL. All solutions were filtered with0.22 μm hydrophobic PVDF membranes (Fisher Scientific) into 10 mmdiameter borosilicate glass tubes and sealed. Solutions were allowed toequilibrate for thirty minutes prior to measuring light scattering.

Individual solutions of α-A, α-B, γD- and γ-S crystallin proteins wereinvestigated to understand the size scale and how the proteins behave indilute solution. (Table 1) Temperature had little effect on protein sizein solution as measured by DLS.

TABLE 1 DLS measurements performed on both modified and unmodified γD-and γ-S- crystallin proteins αA αB γD γS Temp R_(h) R_(h) R_(hf) R_(hs)R_(hf) R_(hs) (° C.) (nm) (nm) (nm) (nm) (nm) (nm)  4° 12 12 3 116 3 10022° 12 13 2.8 104 2.7 102 37° 13 13 2.7 109 2.6 95

Considering that the molecular weight of αA-crystallin is 19.9 kDa andα-B crystalline is 20.16 kDa, the size seen in the DLS experiment isconsistent with α-crystallin's known assembly into 300-1200 kDa speciesin solution as well as in the human eye.

The 3 nm R_(h) for the γ-crystallins represents a single protein specieswhich correlates well with the molecular weights of 20.6 kDa and 20.9kDa for monomeric γD- and γ-S crystallin, respectively. Both speciesalso show aggregates with R_(h) values of 110 and 100 nm for γD- andγS-crystallins, respectively. Aggregation of γ-crystallin has previouslybeen attributed to the attractive interactions between γ-crystallins.The lack of specific protein-protein interactions in the γ-crystallinsallows them to form large aggregates in solution.

γ-crystallins were subjected to a variety of experimental conditions,including a range of pHs (5, 6, 7, 8, 9, 10, 11), concentrations of KCl(100 mM, 150 mM, 300 mM, 500 mM, 1000 mM) and temperature (4.5° C., 22°C., 37° C.). The pH and salt concentration were adjusted via overnightdialysis at 4° C. and their final concentration adjusted to 1.0 g/L.

TABLE 2 Effect of pH and salt on γD- and γ-S crystallin γD γS Rh_(f)Rh_(s) Rh_(f) Rh_(s) Variable (nm) (nm) (nm) (nm) 100 mM NaCl 2.8 1082.7 104 150 mM NaCl 2.8 104 2.7 102 300 mM NaCl 2.6 91 2.9 100 400 mMNaCl 2.7 — 2.6 — 500 mM NaCl 2.8 — 2.5 — 1000 mM NaCl  2.5 — 2.6 — pH 53.9 100 4.8 141 pH 6 2.7 118 2.5 110 pH 8 2.9 102 3.0 96 pH 9 3.5 97 2.9106  pH 10 3.2 — 3.4 90  pH 11 3.1 — 3.1 —

As can be seen in Table 2, varying pH above 10 removed the aggregatesfrom γD- and γS-crystallin solutions. Salt concentrations over 300 mmKCl did reduce γ-crystallin aggregates to individual protein particles.These results indicate that the large aggregates of γ-crystallin can bedisrupted or prevented by interfering with the electrostaticinteractions between the γ-crystallins.

It had been hypothesized that disulfide bonds might mediate the observedγ-crystallin aggregates. The DLS of the γ-crystallin proteins was alsomeasured in 5 mM DTT, and in the presence of 1.0 g/L α-crystallin. Asshown in Table 3, DTT had no effect on the size of the γ-crystallinaggregates. Thus, this hypothesis was incorrect.

Further, the synthesized α-crystallin proteins were mixed withγ-crystallin proteins to determine if the chaperoning ability ofα-crystallin disrupts the γ-crystallin aggregates. The α-A and α-Bcrystallins were each individually mixed with γD- or γS-crystallin in amolar ratio of 3:1, respectively, mimicking the ratio found in the humaneye lens. All solutions were allowed to equilibrate for an hour at 4° C.Upon incubation with α-A or α-B crystallin, the large γ-crystallinaggregate of several hundred nanometers disappeared and the individualγ-crystallin and α-crystallin macromolecules were seen. (Table 3) Thesedata support previous work demonstrating that the α-crystallins suppressnonspecific protein aggregation thus preventing the aggregation ofγ-crystallin proteins.

TABLE 3 Effect of DTT and chaperones on γD- and γ-S crystallin γD γSRh_(f) Rh_(s) Rh_(f) Rh_(s) Additive (nm) (nm) (nm) (nm) — 2.8 104 2.7102 5 mM DTT 2.6 108 3 102 αA 2.7 16 2.8 18 αB 3.2 17 3 19

Without being held to theory, it is hypothesized that the increase insize of α-crystallins (from 63-68 nm to 75 nm) is due to theα-crystallin interaction with denatured or misfolded γ-crystallin. It isclear that the addition of α-crystallin prevents or disrupts the largeγ-crystallin species from forming in solution and by disruptingelectrostatic charges between the γ-crystallins. The α-crystallins areable to disrupt the large aggregates of γ-crystallin that appear at highconcentrations of γ-crystallin. Thus, in the absence of α-crystallin,γ-crystallin will form large soluble aggregates through electrostaticforces that can be interrupted at high pH and high salt concentrations.

Materials and Methods for Characterization of Aggregates

Chemical modification of

D- and

S-crystallin was undertaken to modify the aggregation behavior of theseproteins. The methods used to characterize the modified

D- and

S-crystalline are Matrix Assisted Laser Desorption Ionization-Time ofFlight (MALDI-TOF), circular dichroism, and dynamic light scattering.

Matrix Assisted Laser Desorption Ionization-Time of Flight

Approximately 2 mg of regular or modified γ-crystallin protein wasdialyzed overnight into 5 mM tris (hydroxymethyl) aminomethanehydrochloride (Tris HCl) pH 7. The solution was lyophilized (freezedried) overnight to obtain a dry crystallin protein powder.

Mass spectrometry data were obtained on an Omniflex MALDI-TOF massspectrometer (Bruker Daltonics, Inc., Billerica Mass.) equipped with a337 nm nitrogen laser. Samples (2 mg/mL) were mixed (1:1) with a matrixconsisting of 0.1% trifluoroacetic acid (TFA), 50% acetonitrile and3,5-dimethoxy-4-hydroxycinnamic acid. 1 μL of solution was subsequentlydeposited on a stainless steel target. The instrument was used in linearmode for data acquisition.

Circular Dichroism

Crystallin samples were dialyzed overnight into 10 mM Na₂HPO₄/NaH₂PO₄buffer pH 6.8 and measured at a concentration of 0.5 mg/mL. CD spectrawere measured on a Jasco J715 spectropolarimeter at 22° C. using aquartz cell of 1 mm path length. After allowing the sample toequilibrate for five minutes, spectra were obtained in the range of 250to 195 nm.

Dynamic Light Scattering

Experimental dynamic light scattering (DLS) was measured using an ALVgoniometer instrument which had an ALV-5000/E correlator equipped with288 channels (ALV, Langen Germany) and a 2 W argon laser (Coherent Inc.,Santa Clara, Calif.), with a working power of approximately 40 mW.Scattering intensity was measured at angles between 30° and 90° at 5°intervals, corresponding to a scattering wave vector (q) range between8.41×10⁶ and 2.30×10⁷ m⁻¹. The scattering wave vector is defined asq=4πn sin (θ/2)/λ, where θ is the scattering angle, and λ=514.5, thewavelength of the argon laser in vacuum, and n is 1.33, the refractiveindex of water. The temperature of the sample was held at constanttemperature of 4 to 37±0.1° C. by a circulating water bath.

The correlation function was analyzed using CONTIN analysis to calculatelag times for the correlation functions at measured angles. The peaksrepresent the diffusive mode of the proteins. The delay times (τ) wereconverted to Γ and graphed versus q². The slope of the fitted line isthe diffusion coefficient (D). The lines are fitted such that theintercept is zero because a non-zero value of q=0 is unphysical. The Γversus q² plots were linear with r² values of 0.95 or greater.

DLS measurements were performed on both modified and unmodified γD- and

S-crystallin proteins at 150 mM NaCl, 20 mM Na₂HPO₄/NaH₂PO₄ buffer pH6.8 at a concentration of 0.5 mg/mL. All solutions were filtered with0.22 μm hydrophobic PVDF membranes (Fisher Scientific) into 10 mmdiameter borosilicate glass tubes and sealed. Solutions were allowed toequilibrate for thirty minutes prior to measuring light scattering.

Example 3—Chemical Modification of γD- and γS-Crystalline with PEG24

The first modification of γ-crystallin was done with NHSPEG24. Aminoacids containing primary amines, lysine and arginine, can perform anucleophilic substitution on N-hydroxy succinimide (NHS) functionalizedpoly(ethylene glycol) (PEG). NHS is an activated ester which acceleratesthe Sn2 reaction mechanism because it is a good leaving group. Thenucleophilic substitution produces a protein modified with PEG or aPEGylated

-crystallin protein. PEGylation was chosen because modification ofproteins with PEG has been shown to increase solubility and not affectthe three dimensional structure or properties. In particular, PEG24 waschosen because of its reasonable molecular weight (1100.39) added andspacer arm length (8.82 nm).

Modification of

-crystallin with PEG24 was successful as demonstrated by the largeincrease in

-crystallin molecular weight observed in MALDI-TOF. The maximum relativeintensity for

D-crystallin was at 24,148 m/z or 2 PEG24 units while for

S-crystallin the peak occurred at 26,711 m/z which corresponds to 4PEG24 units. (FIG. 1) The excess reactant and reaction conditions weresufficient as there was no unmodified

-crystallin present in solution. The higher resolution MALDI-TOF data of

D-crystallin shows an additional two distinct peaks at 23,056 m/z and25,206 m/z corresponding to 1 and 3 PEG24 modifications.

CD spectroscopy demonstrated that both crystallin proteins appear tokeep their native state despite modification.

D-crystallin showed increased peaks and depths which could be attributedto a slight difference in protein concentration. The good fit withexperimental data is expected as it has been previously demonstratedthat PEGylation does not interfere with a protein's secondary structure.(data not shown)

DLS was performed on

D- and

S-crystallin modified with PEG24 at 22° C. and 37° C. The NICF provideda distribution function with a single set of peaks which has an angulardependency that can be seen in the graph of Γ versus q². (FIGS. 2-4) ThePEG24 modified

-crystallin protein had a small size distribution with no additionalmode at longer relaxation times which would indicate aggregateformation. For

D-crystallin, the R_(h) calculated from the diffusion coefficient was3.1 nm at 22° and 37° C., while

S-crystallin had an R_(h) of 3.2 nm at 22° and 37° C. The R_(h) valuescorrelated well with the 24.1 kDa and 26.7 kDa molecular weights of

D- and

S-crystallin, respectively, measured by MALDI-TOF.

PEGylation of

-crystallin by NHSPEG24 effectively prevented any aggregation events indilute solutions as evident in DLS. PEG is a hydrophilic polymer and hasbeen shown to increase the solubility of proteins in solution. Withoutbeing held to theory, it is believed that if the aggregation phenomenaare a result of solubility issues associated with hydrophobicinteractions or electrostatics, then the increased solubility associatedwith PEGylation prevents the

-crystallin proteins from aggregation.

Along the same line of thought, without being held to theory, it isbelieved that the overall surface charge of the proteins has beenaltered by the reaction with PEG. At a protein's isoelectric point,there is a decrease in solubility which results from the chargeneutrality associated with the isoelectric point. Amino acids containingprimary amines were the target sites for this type of modification. Theprimary amine of lysine and arginine can have a positive chargeassociated with it depending on the protein makeup and solutionconditions. By having NHSPEG react onto the lysine and arginine groupsthe potentially charged sites were occupied by the hydrophilic PEG,thereby changing the protein surface charge. If the aggregation event isa result of electrostatic interactions or a result of the proteinsproximity to the isoelectric point then this rational would explain whythe aggregate is absent from solution. Modification of

-crystallin protein with PEG via other reaction mechanisms will be usedto examine this possibility.

Without being held to theory, a final explanation for the lack ofaggregates in solution is a spacer issue. In the field of hard sphericalcolloids it has been established that the addition of spacer moleculescan reduce aggregation. Adding the PEG24 moiety to

-crystallin provides a hydrophilic spacer molecule on the surface of theprotein. The spacing between proteins provided by PEG could be all thatis needed to prevent aggregation of

-crystallin protein.

Example 4—Chemical Modification of D- and S-Crystalline with PEG4

-crystallin proteins were modified with PEG4 to investigate the effectof spacer arm length on

-crystallin protein aggregation. The reaction was again performed withNHS functionalized PEG (NHSPEG4) to keep the method of modification thesame. PEG4 has a molecular weight added of 219.33 g/mol and spacer armof 1.6 nm, both of which are smaller than PEG24.

MALDI-TOF data showed an increase in the overall

-crystallin molecular weight indicating modification with PEG4. (FIG. 5)The highest relative intensity seen for

D-crystallin occurred at 22,820 m/z, while for

S-crystallin this occurred at 23,329 m/z which correspond to 4 and 5PEG4 units, respectively, being added to either protein. It should benoted that there is a Gaussian distribution around the relativeintensity peak suggesting that there are proteins which contain both agreater and lesser degree of modification. MALDI-TOF data also showedthat no unmodified crystallin protein is present.

CD spectroscopy again showed that PEGylation did not significantlyaffect the secondary structures of

and α-crystallin proteins. (data not shown) Excellent agreement was seenbetween the native and PEG4 tailored

-crystallin proteins.

The NICF of

-crystallin protein modified with PEG4 had a monoexponetial decay whichindicates a single size scale present in solution. (FIG. 6) Similar to

-crystallin modified with PEG24, no aggregate was present in solution.The distribution function (FIG. 7) again showed a single set of peaksthat demonstrate linear angular dependence in the Γ versus q² graph(FIG. 8). The R_(h) of

D-crystallin measured by DLS was 2.8 and 2.9 nm at 22° C. and 37° C.respectively. The R_(h) size correlates well with the modified proteinweight of nearly 22.8 kDa. A slight increase of the protein monomericsize can also been observed in DLS as the R_(h) of the modified

D-crystallin protein is slightly larger than its unmodified predecessor.The PEG4 modified

S-crystallin protein had an R_(h) of 2.9 at 22° C. and 37° C. Theoverall size of the modified protein did increase in comparison tounmodified

S-crystallin and the R_(h) is consistent with a molecular weight ofapproximately 23.3 kDa.

-crystallin protein modified with NHSPEG4 and NHSPEG24 showed noaggregation at 22° and 37° C. Modification with PEG4 generally resultedin four to five low molecular weight additions while in the case ofPEG24 two to three groups were added per

-crystallin protein. The higher number of modifications made per proteinwith PEG4 provided no significant benefit in preventing aggregation.Similarly, adding a greater total weight with PEG24 to

-crystallin provided no advantage to preventing aggregation.

The

-crystallin proteins modified with PEG24 had a slightly larger R_(h) incomparison to the PEG4 modification. As both modifications resulted inno aggregate it was concluded that spacer arm length does notsignificantly contribute to the prevention of aggregation. It ispredicted that larger PEG chains would produce similar results.

Example 5—Chemical Modification of D- and S-Crystalline with CAPEG

Modification by CAPEG4 was performed to investigate an alternativereaction mechanism for the PEGylation of

-crystallin. At physiological pH (6.8), the primary amine of CAPEG4 isslightly more positive and capable of reacting with the negativelycharged amino acids, aspartic acid and glutamic acid. Alternatively,positively charged amino acids are still capable of reacting with thecarboxylic acid of CAPEG4 (CA). CAPEG4 was selected for similar reasonsas NHSPEG4, those being a small added molecular weight added per unit(265.3 g/mol) and a short spacer arm (1.81 nm).

The MALDI-TOF showed a considerable increase in

-crystallin molecular weight which is the result of modification byCAPEG4. (FIG. 9) There was no unmodified

-crystallin in either solution which suggests that reaction conditionswere sufficient for protein modification. The relative intensity peak at23,273 m/z for

D-crystallin represents 5 CAPEG units having been added to the protein.A higher resolution MALDI-TOF spectrum for

S-crystallin showed several distinct mass peaks to include 22,867 m/z or2 CAPEG4 units, a relative intensity maximum at 23,089 m/z or 3 CAPEG4units, and 23,591 m/z or 5 CAPEG4 units.

The CD spectroscopy of CAPEG4 modification of

-crystallin showed a similar trend to modifications made with PEG4 andPEG24. (Data not shown)

S-crystallin+CAPEG4 showed excellent correlation with the unmodified

S-crystallin protein. The modified

D-crystallin again showed a slight variation at 220 nm but overall thecurve is of a similar shape as the native

D-crystallin.

Modifying

-crystallin with CAPEG also prevented aggregation of proteins. The NICFcan be described by a monoexponetial function which produces adistribution function that has a linear Γ vs q² dependence. (FIG. 10-12)The modified

-crystallin protein was seen in its monomeric form at both 22° and 37°C. where the R_(h) was temperature independent. For

D-crystallin, the diffusion coefficient provided a calculated R_(h) of2.8 nm, while for

S-crystallin this value was 2.9 m. The R_(h) values correlate well withthe 23 kDa and 23.5 kDa modified molecular weights of

D- and

S-crystallin, respectively, in addition to being similar in size to

-crystallin modified with PEG4.

PEGylation with CAPEG at physiological pH (6.8) targeted acidic aminoacids. Similar to the reactions done with NHSPEG, the CAPEG modificationshould alter the overall surface charge of the protein. By reacting withnegatively charaged amino acids the hydrophilic PEG chain occupies thepotentially charged amino acid. Having a similar size and number ofmodifications (four to five) as NHSPEG4,

-crystallin tailored with CAPEG4 offers a very similar modification. Apotential advantage to using CAPEG4 over NHSPEG is that there is no NHSleaving group in the reaction.

Without being held to theory, the CAPEG modification again suggests thatthe protein's surface charge is a key factor to the formation of

-crystallin aggregates. As a PEG moiety is being added to the protein itis also possible that the hydrophobic nature of the surface is altered,preventing aggregation. The crucial information gained from thisexperiment is that modification through acidic or basic amino acids canprevent aggregation.

Example 6—Chemical Modification of D- and S-Crystalline with MMPEG

Maleimide functionalized PEG is yet another reaction mechanism by whichthe

-crystallin proteins may be PEGylated. Thiol groups are capable ofMichael addition over the double bond of the maleimide functional group.Both

-crystallin proteins contain multiple cysteine amino acid residues whichcontain a thiol end group. PEGylation of

-crystallin with MMPEG24 (MM) thus occurs by cysteine amino acids.Modification via this reaction mechanism is unique to previousPEGylation experiments because the overall protein charge should not beaffected. In particular, MMPEG24 was chosen because the high molecularweight added per unit (1239.44 g/mol) and long spacer arm (9.53 nm) willbe comparable with PEG24.

The MALDI-TOF spectra of

-crystallin modified with MMPEG24 showed three distinct peaks can beobserved with minimal to no unmodified

-crystallin. (data not shown) Four

D-crystallin peaks were observed at 23187, 24426, 25666 m/zcorresponding to 1, 2, and 3 modifications. In the case of

S-crystallin peaks were observed at 23565, 24426, and 25666 m/zcorresponding to 1, 2, and 3 modifications. The highest relativeintensity for both crystallin proteins occurred at 2 modifications.

Excellent agreement is again seen between the native and PEGylated

-crystallin proteins. It is known that in some instances the thiolgroups of cysteine are involved in intramolecular disulfide bondingwhich give structural integrity providing the secondary structure. Asboth MM modified

D- and

S-crystallin spectra show a secondary structure similar to native

-crystallin state it was concluded that the modification did not alterthe proteins structure. (data not shown)

PEGylating

-crystallin through its cysteine residues did not prevent aggregation.The NICF curves (data not shown) have an angular dependence whichresulted in the distribution function having two distinct sets of peaks(FIG. 13). The Γ versus q² graph showed two slopes with a linear angulardependence indicating the presence of a slow and fast diffusioncoefficient. (data not shown) At 37° C., the slow mode resulted in anR_(h) of 80 nm for

D-crystallin and 85 nm for

S-crystallin. The large size indicateed that the protein was aggregatingin solution. The fast mode corresponded to monomeric

-crystallin protein, with an R_(h) of 2.8 nm and 3.0 nm for

D- and

S-crystallin at 37° C.

The fact that PEGylation via maleimide functionality did not preventaggregation provides critical information regarding the formation of

-crystallin aggregates. Due to MMPPEG24 and NHSPEG24 both resulting in asimilar number of modifications the surface coverage and spacer groupsaround the

-crystallin proteins must also be similar. The aggregate observed withMMPEG24 modification demonstrates that prevention of aggregation cannotbe not entirely dependent on hydrophilic spacer molecules.

A key insight into the aggregation mechanism is gained in realizing thatPEGylating the protein via uncharged amino acids does not preventaggregation. The key difference between MMPEG and NHSPEG or CAPEG isthat PEGylation via maleimide reaction does not target charged aminoacids of the protein. Without being held to theory, this suggests thatsurface charge and electrostatics play a major role in the formation ofaggregates and must be targeted when disrupting aggregation.

Example 7—Chemical Modification of D- and S-Crystalline with BIOTIN

Biotin is a molecule commonly used for protein modification due to itshigh binding affinity for avidin. Proteins can be tagged with biotin,undergo reactions in vivo or in vitro, then be separated or purifiedfrom the bulk using an avidin column. The NHSBiotin molecule was chosenbecause it reacts through the same mechanism as PEG4 and PEG24 howeverthe added molecule will no longer be hydrophilic but rather contain ahighly charged end group. The molecular weight added per unit is 226.38g/mole with a spacer arm of 1.38 nm.

The

-crystallin proteins were successfully modified with biotin as shown inthe MALDI-TOF. (data not shown)

D-crystallin's peak modification was eight units (23758 m/z) while

S-crystallin's peak occurrence was four units (23325 m/z). Both systemsshow no unmodified

-crystallin indicating reaction conditions were sufficient for thoroughmodification.

The CD spectra showed a sizeable shift upwards as compared with thenative structure of the protein. (data not shown) A shallower well wasobserved around 220 nm which goes against the trend of good correlationbetween modification and secondary structure. The change in spectracould be an indication that the secondary structure of the proteins hasbeen perturbed by the modification. Despite the shift in spectra, theoverall shape of the curve is similar and there was no indication of adisordered or random coil.

Aggregation of

-crystallin protein was observed for

-crystallin modified with biotin. The NICF showed a slow and a fast modewhich resulted in the distribution function having two sets of peaks(FIG. 14). Angular dependence of the distribution function can be seenin the Γ versus q² graph, where a qualitative difference was seenbetween the slow and fast modes. At 22° C., the R_(h) of the fast andslow modes were 2.8 nm and 150 nm for

D-crystallin, while for

S-crystallin these values were 2.6 nm and 180 nm. A slow and fast modewas also observed at 37° C. with the R_(h) values being similar to thoseat 22° C.

The aggregation of

-crystallin modified with NHSBiotin shows that the functionality of themodifying molecule (i.e., the molecular bristle) is important.Mechanistically, biotin modified

-crystallin in the same manner as NHSPEG. Only modifying charged aminoacids of

-crystallin is thus not sufficient in preventing aggregation. Acomparable degree of modification occurred with biotin and PEG4 meaninga similar shift of the protein's isoelectric point. Due to aggregationoccurring in the case of

-crystallin modified with biotin it is not sufficient to purely shiftthe isoelectric point of the

-crystallin protein to prevent aggregation. DLS measurements of thebiotinylated system also further support the notion that aggregationcannot be prevented merely by adding spacer molecules onto the surfaceof

-crystallin.

The stark contrast in chemical properties between PEG and biotin is thereason one prevents aggregation and the other does not. PEG is aflexible hydrophilic molecule whereas the biotin functionally is capableof hydrogen bonding and stabilizing a negative charge. The properties ofPEG thus help the solubility of proteins whereas biotin would contributeto the hydrophobic and electrostatic nature of the aggregates.

Example 8—Chemical Modification of D- and S-Crystalline with BIOTIN-PEG

Modifying

-crystallin proteins with biotin resulted in aggregation so it wasproposed to use NHSBiotinPEG which incorporates a hydrophilic spacerbetween the protein and biotin functionality. It was proposed that thehydrophilic nature of PEG would be the key factor in the prevention of

-crystallin aggregation. The NHS functionalization is used to keep thereaction mechanism constant. The molecular weight added per unit was825.64 g/mol and the spacer arm was 5.6 nm.

An appreciable shift in molecular weight was observed in MALDI-TOFindicating modification of the

-crystallin proteins with BiotinPEG12. (data not shown)

D-crystallin had three distinct peaks observable at 22760, 23582, and24404 m/z corresponding to one, two, and three modifications. Theresolution of

S-crystallin provided MALDI-TOF peaks at 23001, 23816, 24669, 25470, and26122 m/z corresponding to one-five modifications. The MALDI-TOF spectraalso showed no unmodified

-crystallin demonstrating sufficient reaction conditions.

The modified

-crystallin protein's secondary structure was in good agreement withthat of the native

-crystallin as seen in the CD spectra. (data not shown) The wellobserved with CD spectra for

-crystallin modified with just biotin was no longer evident. The PEGportion of the modification most likely allowed for increased solubilityin addition to providing a spacer between the protein and biotinfunctionality.

Despite the incorporation of PEG on the biotin functionality there wasstill aggregate present in solution as can be seen in the slow and fastmode of the NICF. (data not shown) The distribution function (FIG. 15)contained two sets of peaks that demonstrate an angular dependence. TheΓ versus q² graph clearly showed two distinct linear fits, the slope ofwhich provide a slow and fast diffusive mode. (data not shown) The fastdiffusion coefficient corresponds to the monomeric

-crystallin where

D-crystallin had an R_(h) of 2.7 nm at 22° C., while

S-crystallin had the value was 2.6 nm. The slow diffusion coefficientrepresented the large aggregate present in solution with an R_(h) of 105nm for

D-crystallin and 115 nm for

S-crystallin at 22° C. Aggregate of a similar R_(h) was also observed at37° C.

The use of PEG as a spacer between the protein and biotin functionalitydid not aid in preventing aggregation. Without being held to theory andwith reference to the discussion earlier concerning biotin shifting theisoelectric point of

-crystallin, these experimental results also suggest hydrophobics todominate over electrostatics in aggregates forming. The long spacer armof BiotinPEG also did not prevent aggregation giving additional supportto the trend of spacer molecule length not being a crucial factor indeturing aggregation. It should be noted that the

-crystallin tailored with BiotinPEG has a slow mode or aggregate R_(h)which was smaller than the biotin modification suggesting that PEG doesaid in curbing aggregation.

Example 9—Chemical Modification of D- and S-Crystalline withSulfo-N-Hydroxysuccinimide Acetate (SA)

Due to previous modifications with large molecular weights and spacerarms still leading to aggregation, a minimally invasive modification wasstudied. The final modification was done with sulfo-N-hydroxysuccinimideacetate, which incorporated a minimal molecular weight (43 g/mol) andspacer arm (acetate molecule) per unit added. The reaction mechanism issimilar to that of PEG4 so by reacting through the charged amino acidgroups the isoelectric point of

-crystallin should be affected.

A subtle shift in the weight of the

-crystallin proteins was observed in MALDI-TOF. (data not shown) Thepeak relative intensity for

D-crystallin occurred at 22209 m/z and for

S-crystallin at 22562 m/z corresponding to six and seven acetate groups,respectively. The peaks showed no unmodified protein, indicatingsufficient reaction conditions. A Gaussian distribution around the peakrelative intensity indicated that some crystallin proteins have a higherand a lower degree of modification.

A CD spectrum for both modified

-crystallin proteins demonstrated a smaller well around 220 nm ascompared to the native

-crystallin. (data not shown) The change in CD spectra was also observedwith the biotin modification. There is reason to believe that thesecondary structure might be slightly affected by the modificationalthough there are no indications of a random or disordered state.

Looking at the NICF it is evident that modification of

-crystallin with acetate groups did not prevent aggregation. (data notshown) The distribution function is shown in FIG. 16. The angulardependence of the slow and fast diffusive modes can be seen in the Γversus q² graph. (data not shown) For

D-crystallin at 37° C., the fast and slow diffusion coefficientscorrespond to an R_(h) of 2.6 nm and 85 nm. The R_(h) values for

S-crystallin at 37° C. were 2.5 nm and 90 nm. The fast mode R_(h) is anappropriate size for the modified

-crystallin proteins whose molecular weight is approximately 22 kDa.R_(h) values obtained from measurements made at room temperature showedsimilar results.

The SA modification was performed in an attempt to shift the protein'sisoelectric point with a minimal spacer arm. Despite an aggregate beingpresent in solution, the aggregate size was smaller in comparison tounmodified

-crystallin. Through a high number of modifications per protein it ispossible that a substantial shift in isoelectric point did reduce thesize of the aggregate. Without being held to theory, as aggregation isstill present in solution it was concluded that the

-crystallin protein's proximity to the isoelectric point is not solelyresponsible for the aggregation phenomena.

The results of Examples 3-9 are summarized in the following table:

TABLE 4 Summary of results with bifunctional charge masking agents γS γDR_(h) R_(h) R_(h) R_(h) (nm) (nm) (nm) (nm) PEG4 22° C. 2.9 2.8 PEG4 37°C. 2.9 2.9 PEG24 22° C. 3.2 3.1 PEG24 37° C. 3.2 3.1 CAPEG4 22° C. 2.82.9 CAPEG4 37° C. 3 2.8 MMPEG24 22° C. 3.1 100 2.9 85 MMPEG24 37° C. 385 2.8 80 Biotin 22° C. 2.6 180 2.8 150 Biotin 37° C. 2.8 210 2.7 170BiotinPEG 22° C. 2.6 115 2.7 105 BiotinPEG 37° C. 2.8 110 2.6 100 SA 22°C. 2.5 92.5 2.5 82.5 SA 37° C. 2.5 90 2.6 85

The results shown herein show that electrostatics are an importantcomponent of γ-crystallin aggregation. The electrostatic aggregation canbe effectively interrupted using high salt concentrations orbifunctional charge masking agents. A increased understanding of themechanism of γ-crystallin aggregation has provided a new class of agentsthat are particularly useful in the treatment of cataracts andpresbyopia.

Methods: Harvesting and Characterization of Human Cadaver Lenses

Within about 24 hours of death, the eyeball is harvested, sliced and thevitreous is removed. The lens is excised and placed in an incubationmedium called Optisol®. Optisol® is a corneal storage medium containingchondroitin sulfate, dextran 40, optisol base powder, sodiumbicarbonate, gentamycin, amino acids, sodium pyruvate, L-glutamine,2-mercaptoethanol and purified water. OD is the right eye and OS is theleft eye.

Lens opacities are classified according to the LOCS III system. LOCS IIImeasurements are taken with a slit lamp miroscope. The LOCS contains anexpanded set of standards selected from the Longitudinal Study ofCataract slide library at the Center for Clinical Cataract Research,Boston, Mass. It includes six slit lamp images for grading nuclear color(NC) and nuclear opalescence (NO), five retroillumination images forgrading cortical cataract (C), and five retroillumination images forgrading posterior subcapsular (P) cataract. Cataract severity is gradedon a decimal scale, with the standards have regularly spaced intervalson the scale.

Example 10: Testing of CAPEG4 in Human Cadaver Lenses-Transport Acrossthe Epithelium

The transport of the CAPEG4 across an epithelium construct was studied.The epithelium is the outer layer of the cornea and transport across theepithelium has proven to be challenging. The goal was to demonstrate theCAPEG4 construct could be used in a eye drop formulation for transportthrough the front of the eye.

Specifically, the CAPEG4 construct in solution was added to the top ofan apparatus containing culture medium and an epithelium construct.(FIG. 19). The epithelium construct consists of a layer(s) of humancorneal epithelial cells. Aliquots were taken at the top and the bottomof the culture medium from 15 to 120 minutes. FIG. 20 shows the CAPEG4that was transported to the bottom of the cell.

FIG. 21 shows the pH of Opisol® versus the mg/ml of added CAPEG4. Thedata is also presented in tables 5 and 6. Overall, it is demonstratedthat the reduction in coracle and posterior subcapsular cataracts is dueto the CAPEG4 and not a pH effect of the medium. because here was noreduction in corticle cataracts by simply changing (lowering) the pH ofthe Optisol® media with citric acid to the same pH of Optisol®containing the CaPEG amine. Reduction in opacity required the presenceof the active agent.

TABLE 5 Time Top Bottom Min mcg/well Average mcg/well Average % (stock)1 mg/ml CAPEG4 15 427 449 4 13 2.4 472 22 30 448 446 26 31 5.8 445 37 60454 421 8 9 1.7 388 11 45 459 443 9 8 1.5 428 8 90 467 463 17 31 5.7 45945 120 453 481 21 25 4.7 510 30 1000 543 543 mcg/mL Time 0 446 450 4 30.6 harvest: 453 3 16 mg/mL CAPEG4 15 5842 6375 58 60 0.7 6908 62 306099 6312 93 99 1.2 6526 105 60 6450 6972 160 149 1.8 7494 139 45 63136335 677 548 6.7 6356 418 90 6122 6457 525 525 6.4 6792 na 16000 81868186 mcg/mL Blank 7857 7857 1837 1837 23 insret at 15 min

Example 11: Testing of CAPEG4 with Human Cadaver Lenses-Reduction ofCataracts in Human Cadaver Lenses

In all cases, the control is Optisol® medium. The remaining data pointsare for isolated human cadaver lenses treated with CAPEG4 solutions withOptisol®.

Table 6 shows the results for lenses incubated with control medium or 10mg/mL CAPEG4 versus time. In table 6, under the heading cortical, thereis a LOCS III grade of 1.0, 1.0 and 0.9 for a lens incubated with 10mg/ml CAPEG4. The LOCS grades for the control are 1.0, 1.2 and 0.9. At10 mg/mL there was no significant change in the experimental versuscontrol samples.

TABLE 6 LOCS III Grading of cataracts incubated with Optisol ® or 10mg/ml CAPEG4 versus time [CaPEG] Time (mg/mL) (HR) Cortical NO NC PS 10(OD) 0 1.0 5.0 5.0 0.9 Contol (OS) 0 1.2 5.0 5.0 0.9 Optisol Only 10 241.0 5.0 5.0 0.9 Control 24 1.2 5.0 5.0 0.9 Optisol Only 10 72 0.9 5.05.0 0.9 Control 72 0.9 5.0 5.0 1.0

Table 7 shows the results for lenses incubated with control medium or 50mg/mL CAPEG4 versus time. In table 7, under the heading cortical, thereis a LOCS III grade of 1.0 for all samples and a Nuclear opalescence(NO) grade of 2.0 for all samples. There were no significant differencesbetween the controls and 50 mg/mL incubation up to 72 hours for corticalcataracts or for NO. Nuclear color (NC) increased slightly with time forboth the control and upon incubation in the 50 mg/mL CAPEG4 solution.There was a slight improvement in posterior subcapsular cataract from1.1 to 0.9 at o and 24 hours, respectively.

TABLE 7 LOCS III Grading of cataracts incubated with Optisol ® or 50mg/ml CAPEG4 versus time [CaPEG] Time (mg/mL) (HR) Cortical NO NC PS 50(OD) 0 1.0 2.0 1.7 1.1 Contol (OS) 0 1.0 2.0 1.8 0.9 Optisol Only 50 241.0 2.0 1.7 0.9 Control 24 1.0 2.0 1.8 0.9 Optisol Only 50 72 1.0 2.01.9 0.9 Control 72 1.0 2.0 2.0 0.9

Table 8 shows the results for lenses incubated with control medium or100 mg/mL CAPEG4 versus time. There is a decrease in opalescence (NOfrom 1.8 to 1.2) at 0 and 24 hours, respectively for CAPEG4 incubationand minor improvements in cortical and nuclear color up to 72 hours.

TABLE 8 LOCS III Grading of cataracts incubated with Optisol ® or 100mg/ml CAPEG4 versus time [CaPEG] Time (mg/mL) (HR) Cortical NO NC PS 100(OD) 0 3.0 1.8 1.3 0.9 Contol (OS) 0 0.9 1.9 1.5 0.9 Optisol Only 100 242.9 1.2 1.1 0.9 Control 24 0.9 1.8 2.0 0.9 Optisol Only 100 72 2.7 1.21.1 0.9 Control 72 0.9 1.9 2.0 0.9

Table 9 is a repeat of the experiment in Table 8 with different eyes.Under PS (Posterior subcapsular cataracts) the sample incubated at 100mg/ml CAPEG4 had values of 1.8, 1.5, and 1.0 at 0, 24 and 72 hours,respectively. There was also a reduction in cortical cataracts from 3.0to 2.6 to 2.0 at 0, 24, and 72 hours, respectively.

TABLE 9 LOCS III Grading of cataracts incubated with Optisol ® or 100mg/ml CAPEG4 versus time [CaPEG] Time (mg/mL) (HR) Cortical NO NC PS 100(OD) 0 3.0 3.0 3.0 1.8 Contol (OS) 0 1.0 3.0 3.0 1.5 Optisol Only 100 242.6 3.0 3.0 1.5 Control 24 1.0 3.0 3.0 1.5 Optisol Only 100 72 2.0 3.03.2 1.0 Control 72 1.0 3.0 3.1 1.5

Table 10 shows the results for lenses incubated with control medium or200 mg/mL CAPEG4 versus time. Cortical cataracts were reduced from 2.5to 2.2 to 2.0 at 0, 24, and 72 hours, respectively. The control alsoexhibit a slight reduction in cortical cataracts from 3.3 to 3.0 at 24and 72 hours, respectively.

TABLE 10 LOCS III Grading of cataracts incubated with Optisol ® or 200mg/ml CAPEG4 versus time [CaPEG] Time (mg/mL) (HR) Cortical NO NC PS 200(OD) 0 2.5 2.4 2.4 0 Contol (OS) 0 3.3 2.5 2.5 0 Optisol Only 200 24 2.22.4 2.4 0 Control 24 3.3 2.5 2.5 0 Optisol Only 200 72 2.0 2.4 2.4 0Control 72 3.0 2.5 2.5 0

Table 11 shows the results for lenses incubated with control medium or200 mg/mL CAPEG4 versus time. The only decrease in cataract was observedin cortical cataracts for the CAPEG4 solution going from 1.3 to 1.0 to0.9 at 0, 24, and 72 hours, respectively.

TABLE 11 LOCS III Grading of cataracts incubated with Optisol ® or 200mg/ml CAPEG4 (in Optisol ®) versus time [CaPEG] Time (mg/mL) (HR)Cortical NO NC PS 200 (OD) 0 1.3 3.2 2.8 0.9 Contol (OS) 0 1.4 3.2 3.00.9 Optisol Only 200 24 1.0 3.2 2.8 0.9 Control 24 1.4 3.2 3.0 0.9Optisol Only 200 72 0.9 3.2 2.8 0.9 Control 72 1.4 3.2 3.0 0.9

Table 12 shows the results of changing the pH of Optisol® with no addedCAPEG4. The pH was lowered via the addition of citric acid. There was noeffect of changing the pH of the Optisol® on the cataracts.

TABLE 12 Efect of pH of Optisol ® on cataracts Time pH (HR) Cortical NONC PS 6.470 (OD) 0 2.0 5.0 5.2 0.9 6.085 (OD) 0 2.7 2.3 2.0 0.9 6.470 242.0 5.0 5.2 0.9 6.075 24 2.7 2.3 2.0 0.9 6.470 72 2.0 5.0 5.2 0.9 6.07572 2.7 2.3 2.0 0.9

For cadaver lens studies, the different starting point for LOCSIII gradeis due to the varying starting condition in the patients. As theconcentration of the CAPEG4 solutions in Optisol increased, wrinkling ofthe lens capsule was observed and subjectively exhibited some loss oflens volume.

The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item. Theterm “or” means “and/or”. The terms “comprising”, “having”, “including”,and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to”). All ranges disclosed herein areinclusive and combinable.

Embodiments are described herein, including the best modes known to theinventors. Variations of such embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theskilled artisan is expected to employ such variations as appropriate,and the disclosed methods are expected to be practiced otherwise than asspecifically described herein. Accordingly, all modifications andequivalents of the subject matter recited in the claims appended heretoare included to the extent permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed unless otherwise indicated herein or otherwiseclearly contradicted by context.

The invention claimed is:
 1. A method of inhibiting or reversing theprogression of cataract formation, presbyopia, or age relateddegeneration of a crystalline lens in an eye comprising contacting theeye, including the crystalline lens, of a subject in need thereof withan effective cataract-inhibiting amount of an ophthalmic compositioncomprising at least one γ-crystallin charge masking agent, wherein theγ-crystallin charge masking agent is a bifunctional molecule containingNH₂, a succinimide, a carboxylic acid, isocyanate, isothiocyanate,sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride,fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester,epoxide or a fluorophenyl ester, covalently linked to a molecularbristle that is a polyethylene glycol, an alkoxy-polyethylene glycol, oran alkoxypolyethylene glycol having 4 to 200 oxyethylene, alkoxyethyleneor aryloxyethylene groups; poly(2-hydroxypropyl)methacrylamide (HPMA);poly(2-hydroxyethyl)methacrylate (HEMA); a ply(2-oxaziline),poly(m-phosphocholine), poly lysine, or poly glutamic acid, themolecular bristle having a molecular weight of 150 to
 8000. 2. Themethod of claim 1, wherein the γ-crystallin charge masking agent is


3. The method of claim 2, wherein the ophthalmic composition is an eyedrop, a suspension, a gel, an ointment, a spray, or an ophthalmicdevice.
 4. The method of claim 3, wherein the ophthalmic device is acontact lens or a punctal plug.
 5. The method of claim 2, wherein theophthalmic composition is administered by dropping, spraying, injection,iontophoresis or ultrasound enhancement.
 6. The method of claim 2,wherein the ophthalmic composition comprises an amount of the at leastone γ-crystallin charge masking agent that is about 0.001 g to about 0.1g, or about 0.01 g to about 0.05 g.
 7. The method of claim 2, whereinthe ophthalmic composition comprises a buffering agent, an isotonizingagent, a solubilizer, a preservative, a viscosity-increasing agent, achelating agent, an antioxidizing agent, an antibiotic, a sugar, or a pHregulator.
 8. The method of claim 2, wherein the ophthalmic compositionis a sustained release composition.
 9. The method of claim 1, whereinthe ophthalmic composition is an eye drop, a suspension, a gel, anointment, a spray, or an ophthalmic device.
 10. The method of claim 9,wherein the ophthalmic device is a contact lens or a punctal plug. 11.The method of claim 1, wherein the ophthalmic composition isadministered by dropping, spraying, injection, iontophoresis orultrasound enhancement.
 12. The method of claim 1, wherein the molecularbristle is a polyethylene glycol, an alkoxy-polyethylene glycol, or analkoxypolyethylene glycol having 4 to 200 oxyethylene, alkoxyethylene oraryloxyethylene groups.
 13. The method of claim 1, wherein theophthalmic composition comprises an amount of the at least oneγ-crystallin charge masking agent that is about 0.001 g to about 0.1 g,or about 0.01 g to about 0.05 g.
 14. The method of claim 1, wherein theophthalmic composition comprises a buffering agent, an isotonizingagent, a solubilizer, a preservative, a viscosity-increasing agent, achelating agent, an antioxidizing agent, an antibiotic, a sugar, or a pHregulator.
 15. The method of claim 1, wherein the ophthalmic compositionis a sustained release composition.
 16. A method of inhibiting orreversing the progression of cataract formation, presbyopia, or agerelated degeneration of a crystalline lens in an eye comprisingcontacting the eye of a subject in need thereof with an effectivecataract-inhibiting amount of an ophthalmic composition comprising atleast one γ-crystallin charge masking agent, wherein the γ-crystallincharge masking agent is a bifunctional molecule containing NH₂,N-hydroxysuccinimide or COOH covalently linked to a molecular bristlethat is a polyethylene glycol, an alkoxy-polyethylene glycol, or analkoxypolyethylene glycol having 4 to 200 oxyethylene, alkoxyethylene oraryloxyethylene groups; poly(2-hydroxypropyl)methacrylamide (HPMA);poly(2-hydroxyethyl)methacrylate (HEMA); a ply(2-oxaziline),poly(m-phosphocholine), poly lysine, or poly glutamic acid, themolecular bristle having a molecular weight of 150 to
 8000. 17. Themethod of claim 16, wherein the ophthalmic composition is an eye drop, asuspension, a gel, an ointment, a spray, or an ophthalmic device. 18.The method of claim 17, wherein the ophthalmic device is a contact lensor a punctal plug.
 19. The method of claim 16, wherein the ophthalmiccomposition is administered by dropping, spraying, injection,iontophoresis or ultrasound enhancement.
 20. The method of claim 16,wherein the molecular bristle is a polyethylene glycol, analkoxy-polyethylene glycol, or an alkoxypolyethylene glycol having 4 to200 oxyethylene, alkoxyethylene or aryloxyethylene groups.
 21. Themethod of claim 16, wherein the γ-crystallin charge masking agent is


22. The method of claim 16, wherein the ophthalmic composition comprisesan amount of the at least one γ-crystallin charge masking agent that isabout 0.001 g to about 0.1 g, or about 0.01 g to about 0.05 g.
 23. Themethod of claim 16, wherein the ophthalmic composition comprises abuffering agent, an isotonizing agent, a solubilizer, a preservative, aviscosity-increasing agent, a chelating agent, an antioxidizing agent,an antibiotic, a sugar, or a pH regulator.
 24. The method of claim 16,wherein the ophthalmic composition is a sustained release composition.